雷帕霉素对糖尿病肾病大鼠足细胞生物学行为及mTOR信号通路的影响

doi:10.19586/j.2095-2341.2021.0169

生物技术进展 ›› 2022, Vol. 12 ›› Issue (3): 460-466.DOI: 10.19586/j.2095-2341.2021.0169

雷帕霉素对糖尿病肾病大鼠足细胞生物学行为及mTOR信号通路的影响

黄乔木¹(), 何艳²()

^1.咸宁市中心医院/湖北科技学院附属第一医院肾内科，湖北咸宁 437199
^2.咸宁市第一人民医院重症医学科，湖北咸宁 437099

收稿日期:2021-10-20 接受日期:2021-11-12 出版日期:2022-05-25 发布日期:2022-05-26
通讯作者: 何艳
作者简介:黄乔木 E-mail：dk8922xl@163.com；
基金资助:
湖北省卫生健康委员会2019—2020年度创新团队项目(WJ2019M096)

Effects of Rapamycin on the Biological Behaviors of Podocytes and mTOR Signaling Pathway in Rats with Diabetic Nephropathy

Qiaomu HUANG¹(), Yan HE²()

^1.Department of Nephrology，Xianning Central Hospital/The First Affiliated Hospital of Hubei，University of Science and Technology，Hubei Xianning 437199，China
^2.Department of Critical Care Medicine，the First People's Hospital of Xianning，Hubei Xianning 437099，China

Received:2021-10-20 Accepted:2021-11-12 Online:2022-05-25 Published:2022-05-26
Contact: Yan HE

摘要/Abstract

摘要：

为了探究雷帕霉素对糖尿病肾病大鼠足细胞生物学行为及哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）信号通路的影响，采用链脲霉素腹腔注射构建糖尿病肾病大鼠模型，将正常大鼠体内取出的足细胞设为对照组，模型大鼠体内取出的足细胞设为糖尿病肾病模型组（DN组），取2 mg·kg^-1雷帕霉素干预DN组足细胞，并将其设为雷帕霉素组（RAPA组）。采用3-（4，5-二甲基噻唑-2）-2，5-二苯基四氮唑溴盐[3-（4，5-dimethylthiahiazo-z-y1）-2，5-diphenytetrazoliumromide，MTT]法检测足细胞增殖水平，Transwell检测细胞迁移和侵袭能力，流式细胞术检测细胞凋亡水平，Western blot法检测上皮-间充质转化标志物[E-钙黏蛋白（E-cadherin）、N-钙黏蛋白（N-cadherin）、波形纤维蛋白（vimentin）]、mTOR和核糖体S6激酶1（S6K1）蛋白表达水平。结果显示，与对照组相比，DN组细胞增殖水平显著被抑制，细胞迁移、侵袭水平显著升高，细胞凋亡率显著增加，上皮-间充转标志物E-cadherin表达显著下调，N-cadherin和Vimentin表达显著上调，mTOR/S6K1信号通路被显著活化（P<0.05）。与DN组相比，RAPA组细胞增殖水平显著升高，细胞迁移、侵袭水平显著降低，细胞凋亡率显著降低，E-cadherin表达显著上调，N-cadherin和Vimentin表达显著下调，mTOR和S6K1的蛋白表达显著被抑制（P<0.05）。结果表明，雷帕霉素通过抑制mTOR信号通路，促进足细胞体外增殖，抑制细胞迁移、侵袭、凋亡和上皮-间充质转化，发挥对糖尿病肾病大鼠足细胞的保护作用。

关键词: 雷帕霉素, 糖尿病肾病, 足细胞, 细胞生物学行为, mTOR信号通路

Abstract:

To explore the effects of rapamycin on the biological behaviors of podocytes and mammalian target of rapamycin （mTOR） signaling pathway in rats with diabetic nephropathy， streptozotocin was injected intraperitoneally to construct a rat model of diabetic nephropathy. The podocytes taken from normal rats were set as control group， and the podocytes taken from the model rats were enrolled as diabetic nephropathy model group （DN）. The podocytes taken from DN group were intervened with 2 mg·kg^-1 rapamycin and selected as rapamycin group （RAPA）. 3-（4， 5-dimethylthiahiazo-z-y1）-2， 5-diphenytetrazoliumromide （MTT） was used to detect podocyte proliferation level， and Transwell was used for detection of cell migration and invasion and flow cytometry for cell apoptosis level. Western blot was adopted to measure the protein expression levels of epithelial-mesenchymal transition markers （E-cadherin， N-cadherin， vimentin）， mTOR and ribosomal protein S6 kinase 1 （S6K1）. The results showed that， compared to the control group， the cell proliferation level was significantly inhibited， the cell migration and invasion were significantly increased， the apoptosis rate was significantly enhanced， the expression of epithelial-mesenchymal transition marker E-cadherin was significantly down-regulated while the expressions of N-cadherin and Vimentin were significantly up-regulated， the mTOR/S6K1 signaling pathways were significantly activated in the DN group（P<0.05）. Compared to the DN group， the cell proliferation level was significantly increased， the cell migration and invasion in the RAPA group were significantly reduced， the apoptosis rate was significantly reduced， the expression of E-cadherin was significantly up-regulated while the expressions of N-cadherin and Vimentin were significantly down-regulated， the protein expressions of mTOR and S6K1 were significantly inhibited in the RAPA group （P<0.05）. The results indicated that rapamycin could promote the proliferation of podocytes in vitro and inhibit the cell migration， invasion， apoptosis and epithelial-mesenchymal transition by inhibiting the mTOR signaling pathway， and it exerts a protective effect on podocytes in rats with diabetic nephropathy.

Key words: rapamycin, diabetic nephropathy, podocytes, cell biological behaviors, mTOR signaling pathway

中图分类号:

R587.2

黄乔木, 何艳. 雷帕霉素对糖尿病肾病大鼠足细胞生物学行为及mTOR信号通路的影响[J]. 生物技术进展, 2022, 12(3): 460-466.

Qiaomu HUANG, Yan HE. Effects of Rapamycin on the Biological Behaviors of Podocytes and mTOR Signaling Pathway in Rats with Diabetic Nephropathy[J]. Current Biotechnology, 2022, 12(3): 460-466.

图/表 6

图1 各组足细胞形态学变化注：放大倍数为200倍。

Fig.1 Morphological changes of podocytes in each group

图2 各组足细胞增殖水平比较注：**表示与对照组相比，差异具有统计学意义（P<0.01）；#表示与DN组相比，差异具有统计学意义（P<0.05）

Fig.2 Comparison of podocyte proliferation level in each group

图3 各组足细胞迁移、侵袭水平比较A：Transwell实验中以结晶紫染色后迁移、侵袭的细胞；B：Transwell实验中迁移、侵袭细胞定量结果。***表示与对照组相比，差异具有统计学意义（P<0.001）；###表示与DN组相比，差异具有统计学意义（P<0.001）

Fig.3 Comparison of podocyte migration and invasion in each group

图4 各组足细胞上皮?间充质转化水平差异A：E?cadherin、N?cadherin、Vimentin蛋白条带灰度值；B：E-cadherin、N-cadherin、Vimentin蛋白表达定量结果。***表示与对照组相比，差异具有统计学意义（P<0.001）；###表示与DN组相比，差异具有统计学意义（P<0.001）

Fig.4 Comparison of podocyte epithelial?mesenchymal transition in each group

图5 各组足细胞凋亡水平比较A：流式细胞仪检测细胞凋亡染色图；B：流式细胞术检测细胞凋亡率。***表示与对照组相比，差异具有统计学意义（P<0.001）；###表示与DN组相比，差异具有统计学意义（P<0.001）

Fig.5 Comparison of podocyte apoptosis in each group

图6 各组足细胞内mTOR信号通路表达比较A：mTOR、S6K1蛋白条带灰度值；B：mTOR、S6K1蛋白表达定量。***表示与对照组相比，差异具有统计学意义（P<0.001）；##表示与DN组相比，差异具有统计学意义（P<0.01）；###表示与DN组相比，差异具有统计学意义（P<0.001）

Fig.6 Comparison of mTOR signaling pathway in podocytes of each group

参考文献 20

1	TESCH G H. Diabetic nephropathy-is this an immune disorder[J]. Clin. Sci. (Lond), 2017, 131(16): 2183-2199.
2	PERKOVIC V, JARDINE M J, NEAL B, et al.. Canagliflozin and renal outcomes in type 2 diabetes and nephropathy[J]. N. Engl. J. Med., 2019, 380(24): 2295-2306.
3	VLAD A, VLAD M, PETRICA L, et al.. Therapy with atorvastatin versus rosuvastatin reduces urinary podocytes,podocyte-associated molecules,and proximal tubule dysfunction biomarkers in patients with type 2 diabetes mellitus:a pilot study[J]. Ren. Fail., 2017, 39(1): 112-119.
4	SCHNITZBAUER A A, FILMANN N, ADAM R, et al.. mTOR Inhibition is most beneficial after liver transplantation for hepatocellular carcinoma in patients with active tumors[J]. Ann. Surg., 2020, 272(5): 855-862.
5	SUN S Y. mTOR-targeted cancer therapy: great target but disappointing clinical outcomes,why[J]. Front Med., 2021, 15(2): 221-231.
6	ZHAO X F, LIAO Y, ALAM M M, et al.. Microglial mTOR is neuronal protective and antiepileptogenic in the pilocarpine model of temporal lobe epilepsy[J]. J. Neurosci., 2020, 40(40): 7593-7608.
7	KON N, OU Y, WANG S J, et al.. mTOR inhibition acts as an unexpected checkpoint in p53-mediated tumor suppression[J]. Genes Dev., 2021, 35(1-2): 59-64.
8	顾杰,张春江,贾林,等.大鼠足细胞的原代培养及其Nephrin的特异表达[J].广东医学,2017,38(6):825-829.
9	KE R, WANG Y, HONG S, et al.. Endoplasmic reticulum stress related factor IRE1α regulates TXNIP/NLRP3-mediated pyroptosis in diabetic nephropathy[J/OL]. Exp. Cell Res., 2020, 396(2): 112293[2022-01-06]. .
10	JIANG N, ZHAO H, HAN Y, et al.. HIF-1α ameliorates tubular injury in diabetic nephropathy via HO-1-mediated control of mitochondrial dynamics[J/OL]. Cell Prolif.,2020,53(11): e12909[2022-01-06]. .
11	LI Y, PAN Y, CAO S, et al.. Podocyte EGFR inhibits autophagy through upregulation of rubicon in type 2 diabetic nephropathy[J]. Diabetes., 2021, 70(2): 562-576.
12	高婧雅,郭庆军,谢炎,等.雷帕霉素联合索拉非尼在超米兰标准肝移植肝细胞癌复发患者中的疗效观察[J].中国普外基础与临床杂志,2018,25(9):1039-1043.
13	UNNO R, KAWABATA T, TAGUCHI K, et al.. Deregulated MTOR (mechanistic target of rapamycin kinase) is responsible for autophagy defects exacerbating kidney stone development[J]. Autophagy, 2020, 16(4): 709-723.
14	GAO G, CHEN W, YAN M, et al.. Rapamycin regulates the balance between cardiomyocyte apoptosis and autophagy in chronic heart failure by inhibiting mTOR signaling[J]. Int. J. Mol. Med., 2020, 45(1): 195-209.
15	LIU L, YANG L, CHANG B, et al.. The protective effects of rapamycin on cell autophagy in the renal tissues of rats with diabetic nephropathy via mTOR-S6K1-LC3II signaling pathway[J]. Ren. Fail, 2018, 40(1): 492-497.
16	LI M Y, ZHU X L, ZHAO B X, et al.. Adrenomedullin alleviates the pyroptosis of leydig cells by promoting autophagy via the ROS-AMPK-mTOR axis[J/OL]. Cell Death Dis., 2019, 10(7): 489[2022-01-06]. .
17	HOU Y, WANG K, WAN W,et al.. Resveratrol provides neuroprotection by regulating the JAK2/STAT3/PI3K/AKT/mTOR pathway after stroke in rats[J]. Genes Dis., 2018, 5(3): 245-255.
18	葛超冉,王鑫,钟赣生,等.海藻玉壶汤及拆方对甲状腺肿大模型大鼠mTOR-p70S6K/4E-BP1信号通路的影响[J]. 北京中医药大学学报,2021,44(5):436-443.
19	KASAHARA K. Physiological function of FKBP12,a primary target of rapamycin/FK506:a newly identified role in transcription of ribosomal protein genes in yeast[J]. Curr. Genet., 2021,67(3): 383-388.
20	BONUCCI M, KUPERWASSER N, BARBE S, et al.. mTOR and S 6K1 drive polycystic kidney by the control of Afadin-dependent oriented cell division[J/OL]. Nat. Commun., 2020, 11(1): 3200[2022-01-06]. .

[1]	王珍,杨洛,廖敏,郝亚荣. mTOR信号通路在糖尿病肾病发病机制中的研究进展[J]. 生物技术进展, 2021, 11(3): 316-321.
[2]	金童,陈铖. 内质网应激及其在糖尿病肾病中的作用机制[J]. 生物技术进展, 2021, 11(1): 40-46.

雷帕霉素对糖尿病肾病大鼠足细胞生物学行为及mTOR信号通路的影响

Effects of Rapamycin on the Biological Behaviors of Podocytes and mTOR Signaling Pathway in Rats with Diabetic Nephropathy

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 20

相关文章 2

编辑推荐

Metrics

本文评价